Chromatographic analysis

ABSTRACT

A method for the microdetermination of 2,8-dioxyadenine in whole blood plasma by strong acid cation exchange resin chromatography with a linear or stepwise gradient increase in concentration of HC1 for elution.

United States Patent Inventors Appl. No. Filed Patented Assignee CHROMATOGRAPHIC ANALYSIS 12 Claims, No Drawings US. (I 23/230 B, 23/230 R, 23/253 R, 74/61.1 C, 356/39 Int. (1 ....G01n2 l/00', G0ln 31/04, G01n 33/16 Field ofSearch 23/230 B, 230, 253; 356/223, 224, 39; 73/611; 260/21 1.5; 250/7l.5

[56 References Cited UNITED STATES PATENTS 3,157,635 11/1964 Tanaka et al. 260/21 1.5 3,414,383 12/1968 Murphy 23/230 Primary Examiner-Morris O. Wolk Assistant Examiner-R. E. Serwin Attorneys-Walter C. Kehm and Scott J. Meyer ABSTRACT: A method for the microdetermination of 2,8- dioxyadenine in whole blood plasma by strong acid cation exchange resin chromatography with a linear or stepwise gradient increase in concentration of HCl for elution.

cnnomx'roomnrc ANALYSIS This invention relates to a method for the determination of 2,8-dioxyadenine and more particularly to the chromatographic microdetermination of 2,8-dioxyadenine in whole blood plasma or serum.

Adenine, the precursor of 2,8-dioxyadenine, is an important purine base constituent of nucleic acid and plays an essential role in the life cycle. Adenine was first discovered by Kossel and was subsequently synthesized and its molecular structure confirmed by Emil Fischer, U.S. Pat. No. 607,029.

Despite the long-recognized presence of adenine compounds in living cells, it is still not clear how much, if any, of adenine present in the diet in various combined forms enters into'the bloodstream as free adenine. However, small quantities, on the order of about 1 milligram per day, of adenine have been reported to be present as a normal component of fasting human urine.

Irrespective of whether adenine is a normal metabolic intermediate, it is known that exogenous adenine can be readily metabolized by man and various mammalian species. The incorporation of adenine into animal tissues after oral, intravenous or intraperitoneal administration has been reported by a number of investigators. It has also been reported that oral or intraperitoneal administration of large doses of adenine in various animals leads to the deposition in the kidney of a compound which was originally thought to be uric acid but later proved to be 2,8-dioxyadenine. Bendich et al., J. Biol. Chem, 183, 267-77 (1950). The structures of the latter compound and its precursor, adenine, are shown below for comparison:

Through the discovery of Simon and Finch, J. Clin. Invest, 41, 35l-9 (1962), 44, 629-42 (1965) and Blood, 20, 485-91 (1962), it was learned that the addition to blood of a very small amount of adenine (0.75125 micromoles per milliliter) at the beginning of storage in ACD (acid-citrate-dextrose) could maintain higher levels of ATP in the erythrocytes and improve the survival of the red cell. Much evidence has now been accumulated which supports the desirable use of adenine as a blood preservative and its use in blood banking of ACD blood. Such preservation of blood with adenine and other purine derivatives, especially inosine and guanosine, is described by Nakao et al., Japan Pat. No. 17,582 (1962) and by Fischer et al., Bibliotheca Haemat., l2, 77-91 (I961).

In view of the utility of adenine in blood preservation and the known fonnation of small amounts of undesirable 2,8- dioxyadenine under various conditions, a convenient and reliable method for the determination of the latter compound in blood plasma would be of much use.

Accordingly, it is an object of this invention to provide a method for the determination of 2,8-dioxyadenine in whole blood plasma.

It is another object of this invention to provide a method for the chromatographic microdetermination of 2,8-dioxyadenine in whole blood plasma or serum without deproteinization.

Other objects and advantages of the invention described herein will be apparent to those skilled in the art.

In brief, the method of this invention comprises the microdetermination of 2,8-dioxyadenine in whole blood plasma or serum by strong acid cation exchange resin chromatography employing a linear or stepwise gradient increase in concentration of strong acid for elution.

It is known that purine bases form cations in strongly acid solutions. Thus, the ionic species of adenine in strong acid is These cations can be absorbed on a cation exchange resin, such as the sulfonic acid type containing acidic SO; groups, and permit the separation of basic purines from biological fluids by cation exchange chromatography. This general principal for the separation of purines is described by Bergmann and Dikstein, New Methods of Purification and Separation of Purines" in Methods of Biochemical Analysis," Vol. 6, Glick, Ed., lnterscience Publishers, lnc., New York, 1958, pp. 86-87. The use of such sulfonic acid cation exchangers and other ion exchange resins for the separation of purines, and related nitrogenous organic compounds is further described in U.S. Pat. Nos. 2,891,945 and 3,157,635.

In the determination of purines and purine derivatives in blood plasma, in general, it has heretofore been thought to be necessary to deproteinize the plasma prior to removing selectively the purines present in solution. Bergmann and Dikstein, "New Methods for Purification and Separation of Purines, supra, at page 93; Wyngaarden, Arch. Biochem. Biophys., 70, -6 (1957); Busch and von Borcke, Nature, 210, 631 (1966). It has now unexpectedly been found that even a microdetermination of the compound 2,8-dioxyadenine can be performed in whole plasma that has not been subjected to deproteinization by the employment of the method of this invention. Moreover, the analytical determination is substantially improved according to the method of this invention since it has been found that deproteinization results in losses of about 50 percent of the 2,8-dioxyadenine in the plasma sample. It has also been found that the chromatographic columns used according to this method can be used repeatedly (e.g., five times) after merely washing with water instead of the conventional recycling treatment that is required, in general, in ion exchange chromatography. The improved analytical results, the consequent savings in time and the convenience of this method represent outstanding advantages of the invention.

By the term microdetermination" is meant the determination of quantities of 2,8-dioxyadenine in blood plasma as little as l to 15 micrograms per milliliter. These quantities are of a much smaller magnitude than the approximately 150 pg/ml. quantities that may be present in urine after administration of large amounts of adenine. .It was unexpected that such small quantities of 2,8-dioxyadenine could be determined in blood plasma without extensive prior treatment of the plasma such as by deproteinization with trichloroacetic acid or tungstic acid, extraction with l-lCl0,, and the like procedures since such procedures are generally required as noted above.

Illustrative of the strong acid cation exchange resins which can be employed in the practice of this invention are the sulfonic acid cation exchange resins. These resins can be, for example, sulfonated phenol, sulfonated polystyrene, sulfonated phenolformaldehyde, sulfonated copolymers of monoalkenyl aromatic hydrocarbons with from about 0.5 to about 25 weight percent of a dialkenyl cross-linking compound, e.g., divinylbenzene, divinyltoluene, divinylxylene, and the like resins. Resins of this type are commercially available under the trademarks Dowex 50" (Dow Chemical Co.), Amberlite lR-l20 (Rohm & Haas Co., Inc.), "Driolite C-3" (Chemical Process Co., Inc.), and Diaion SKOl (Mitsubishi Chemical Industries, Ltd.). A preferred resin is Dowex 50" which is a sulfonated polystyrene cross linked with divinylbenzene such as described in US. Pat. No. 2,366,007.

Other suitable strong acid cation exchange resins which can be used in this invention will be apparent to those skilled in the art and can be prepared by reference to a text such as Kunin, lon Exchange Resin, John Wiley & Sons, Inc., New York, 1950.

In carrying out the method of this invention, a linear gradient increase from to about 4N HCl can be used, but it is preferred to use a stepwise gradient increase in concentration of HCl performed in three steps, from IN to 2N to 4N HCl. After applying the blood plasma to the cation exchange resin column, washing with water followed by 1N l-lCl removes proteins and various other blood constituents. 2,8- Dioxyadenine is then removed by eluting with 2N HCl. By increasing the HCl concentration to 4N, adenine is eluted.

A suitable chromatographic column in the present method can comprise, for example, resin bed dimensions of about cm. long and 0.9 cm. diameter. The resin bed height-to-width ratio can be varied from about 5:1 to about 20:1, with the flow rate preferably maintained at below about 0.75 ml. per minute. By employing such a column, up to about 25 ml. of plasma can be analyzed on a column of 16 ml. resin bed volume. Other suitable resin bed dimensions can be readily determined by those skilled in the art and it will be understood that the invention is not limited to the specific dimensions described above.

In the eluate fractions, 2,8-dioxyadenine is determined optically by (1) ultraviolet spectrophotometry at about 305 mg or by (2) fluorescence spectrometry at about 540 my. with activation at 495 my after reaction at about neutral pH with dichloroquinone chlorimide (DOC). While it has been previously known that 2,8-dioxyadenine has characteristic ultraviolet absorption spectra with a maximum at 305 mg, the identification by fluorescence spectrometry with DQC was unexpected since it is known that neither xanthine nor hypoxanthine,

which are close analogs of 2,8-dioxyadenine, form fluorescent derivatives with DQC.

The ion exchange resin column used in the present invention can be readily adapted to placement in small glass or flexible plastic tubes which can be made available to doctors, clinical laboratories and hospitals for convenient determination of 2,8-dioxyadenine in blood plasma samples with a minimum of setup equipment. Examples of such tubes and their adaptation for ion exchange resins are described in U.S. Pat. Nos. 2,702,034; 3,206,602; 3,305,446; and 3,464,798.

The following examples will further illustrate the present invention although it will be understood that the invention is not limited to these specific examples.

EXAMPLE 1 sample of whole blood plasma preserved with ACD and containing 1 to 15 ,ug. of 2,8-dioxyadenine and pg. of adenine per ml. exogenously added to the plasma is adjusted to pH 6.0 and 25 ml. are passed through the column at a flowrrate of about 0.75 ml. per minute. Absorbed proteins are removed from the column by washing with 25 ml. of water and additional interfering substances are eluted with 20 ml. of 1N HCl. The BC! concentration is then increased to 2N, and five frac-' tions of 5 ml. each are collected to elute 2,8-dioxyadenine. After increasing the HCl concentration to 4N, five fractions of 5 ml. each are collected to obtain adenine.

The amount of 2,8-dioxyadenine in the eluants collected at 2N HCl is quantitated by ultraviolet spectrophotometry at 305 mp. and by fluorescence spectrometry with DQC at 540 mp. after activation at about 495 mp. In the latter procedure, the eluant is first neutralized to pH 7.5 with K,CO,. To 2.0 ml. of the neutralized eluant is added 0.1 ml. of ethanolic DQC reagent (1 mg./ml.). After 20 minutes, the solution is extracted with 1.0 ml. of ethyl acetate, and the aqueous layer examined in a spectrophotofluorometer or fluorometer for fluorescence.

Using the above procedures in typical runs, from about I to about 15 pg. of 2,8-dioxyadenine per ml. of blood plasma were detected. At these concentrations of 2,8-dioxyadenine, the 2N HCl eluate fractions are preferably pooled.

EXAMPLE 2 The procedure of example 1 is repeated except that a resin bed of Amberlite lR-l20 is substituted for the Dowex 50W-X8 resin bed. Substantially similar detection of 2,8- dioxyadenine in blood plasma samples is obtained.

EXAMPLE 3 The procedure of example 1 is repeated except that l) citrated and (2) heparinized blood plasma, respectively, are substituted for the ACD blood plasma. Substantially similar detection of 2,8-dioxyadenine in blood plasma samples is obtained.

EXAMPLE 4 The procedure of example 1 is repeated, but with deproteinized plasma and again with whole plasma in order to show the importance of omitting the conventional deproteinizing step. The deproteinizing was accomplished with tungstic acid according to the procedure described by Kolmer et al., Approved Laboratory Technic," 5th ed. l, published by Appleton-Century-Crofts Inc., New York, at page 965. In typical runs, recoveries of 2,8-dioxyadenine from the deproteinized plasma samples were only 54 percent of those for the whole plasma samples. About 10 minutes of time were saved in each run by omitting the deproteinization STEP.

Various other examples and modifications of the foregoing examples will be apparent to the person skilled in the art after reading the foregoing description and appended claims without departing from the spirit and scope of the invention. For example, it will be appreciated that an equivalent amount of another strong acid, e.g., l-l,S0 can be substituted for BC! in the above examples with substantially similar results. All

such further examples and modifications of the foregoing examples are included within the scope of the invention as defined in the appended claims.

What is claimed is:

l. A process for the microdetermination of 2,8-dioxyadenine in blood plasma or serum without deproteinization which comprises contacting said blood plasma or serum with a column of strong acid cation exchange resin, applying to the column a gradient increase in concentration of strong acid and determining the 2,8-dioxyadenine in the eluant fractions by ultraviolet spectrophotometry at about 305 my" 2. The process of claim 1 in which a stepwise gradient increase in concentration of HCl is employed in three steps, from IN to 2N to 4N HCl and the 2,8-dioxyadenine is determined in the 2N eluant fractions.

3. The process of claim 1 in which the strong acid cation exchange resin is a sulfonic acid type resin.

4. The process of claim 1 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene.

5. The process of claim 1 in which the resin bed height to width ratio ranges from about 5:1 to about :1, and the flow rate is maintained at below about 0.75 ml. per minute.

6. The process of claim 2 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene, the resin bed height to width ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute.

7. A process for the microdetermination of 2,8-dioxyadenine in blood plasma or serum without deproteinization which comprises contacting said blood plasma or serum with a column of strong acid cation exchange resin, applying to the column a gradient increase in concentration of strong acid an determining the 2,8-dioxyadenine in the eluant fractions by fluorescence spectrometry at about 540 my. [with activation at 495 my. after reaction at about neutral pH with dichloroquinone chlorimide.

8. The process of claim 7 in which a stepwise gradient increasein concentration of HCl is employed in three steps, from lN to 2N to 4N HCl and the 2,8-dioxyadenine is determined in the 2N eluant fractions.

9. The process of claim 7 in which the strong acid cation exchange resin is a sulfonic acid type resin.

10. The process of claim 7 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene.

11. The process of claim 7 in which the resin bed height-towidth ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute.

12. The process of claim 8 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene, the resin bed height-to-width to width ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute. 

2. The process of claim 1 in which a stepwise gradient increase in concentration of HC1 is employed in three steps, from 1N to 2N to 4N HC1 and the 2,8-dioxyadenine is determined in the 2N eluant fractions.
 3. The process of claim 1 in which the strong acid cation exchange resin is a sulfonic acid type resin.
 4. The process of claim 1 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene.
 5. The process of claim 1 in which the resin bed height to width ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute.
 6. The process of claim 2 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene, the resin bed height to width ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute.
 7. A process for the microdetermination of 2,8-dioxyadenine in blood plasma or serum without deproteinization which comprises contacting said blood plasma or serum with a column of strong acid cation exchange resin, applying to the column a gradient increase in concentration of strong acid an determining the 2,8-dioxyadenine in the eluant fractions by fluorescence spectrometry at about 540 m Mu (with activation at 495 m Mu after reaction at about neutral pH with dichloroquinone chlorimide.
 8. The process of claim 7 in which a stepwise gradient increase in concentration of HCl is employed in three steps, from 1N to 2N to 4N HCl and the 2,8-dioxyadenine is determined in the 2N eluant fractions.
 9. The process of claim 7 in which the strong acid cation exchange resin is a sulfonic acid type resin.
 10. The process of claim 7 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene.
 11. The process of claim 7 in which the resin bed height-to-width ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute.
 12. The process of claim 8 in which the cation exchange resin is a polystyrene-sulfonic acid resin cross linked with divinylbenzene, the resin bed height-to-width to width ratio ranges from about 5:1 to about 20:1, and the flow rate is maintained at below about 0.75 ml. per minute. 